Development of a novel output value for quantitative assessment in methylated DNA immunoprecipitation-CpG island microarray analysis

Abstract

In DNA methylation microarray analysis, quantitative assessment of intermediate methylation levels in samples with various global methylation levels is still difficult. Here, specifically for methylated DNA immunoprecipitation-CpG island (CGI) microarray analysis, we developed a new output value. The signal log ratio reflected the global methylation levels, but had only moderate linear correlation (r = 0.72) with the fraction of DNA molecules immunoprecipitated. By multiplying the signal log ratio using a coefficient obtained from the probability value that took account of signals in neighbouring probes, its linearity was markedly improved (r = 0.94). The new output value, Me value, reflected the global methylation level, had a strong correlation also with the fraction of methylated CpG sites obtained by bisulphite sequencing (r = 0.88), and had an accuracy of 71.8 and 83.8% in detecting completely methylated and unmethylated CGIs. Analysis of gastric cancer cell lines using the Me value showed that methylation of CGIs in promoters and gene bodies was associated with low and high, respectively, gene expression. The degree of demethylation of promoter CGIs after 5-aza-2'-deoxycytidine treatment had no association with that of induction of gene expression. The Me value was considered to be useful for analysis of intermediate methylation levels of CGIs.

Figure 1

Distribution of methylation levels assessed by the available output values and the Me value in a gastric cancer cell line, AGS, and the same cell line after 5-aza-dC treatment. The analysis was performed for the signal log ratio with background subtraction normalization (A), the P[X] value (B), the P value (C), and the Me value (D). Lines between the two bar graphs indicate values of individual probes (randomly selected representative 20 values). Distributions of the signal log ratio and the Me value reflected the global methylation level, as shown by their shift towards smaller values in cells with 5-aza-dC treatment. In contrast, those of the P[X] and P values did not show the shift.

Figure 2

Correlation between an output value and the fraction of methylated CpG sites by bisulphite sequencing. Forty samples with variable methylation levels (11 loci in four cell lines) were analysed by bisulphite sequencing, and the fraction of methylated CpG sites among all the CpG sites within 200 bp of the microarray probes was obtained. The numbers of CpG sites in this region ranged from 7 to 21. The Me value had a linear correlation with the fraction (r = 0.88, Pearson's correlation coefficient).

Figure 3

Association between DNA methylation level and gene expression. (A) Average methylation levels (site Me values) of genes with high and low expression. Genes with low expression (dashed line) showed significantly higher methylation levels than those with high expression (solid line) in positions close to TSSs. (B) Gene expression levels according to methylation statuses of the promoters (left panel) and gene bodies (right panel). Genes were classified into unmethylated (CGI Me value <0.4, dotted line), moderately methylated (0.4 ≤ CGI Me value ≤ 0.6, dashed line), and highly methylated (CGI Me value >0.6, solid line). Methylation of promoters was strongly associated with low expression (P = 8 × 10⁻²⁰³ in AGS, t-test of expression of methylated genes and unmethylated genes), and methylation of CGIs in gene bodies was associated with higher expression (P = 6 × 10⁻⁴ in AGS).

Figure 4

Degree of demethylation according to the positions against TSSs, and the lack of association between the degree of demethylation and that of re-expression. (A) Degree of demethylation according to the positions against TSSs. Methylation levels were analysed for CGIs methylated in AGS without 5-aza-dC treatment. 5-Aza-dC treatment induced demethylation of upstream CGIs, gene body CGIs, downstream CGIs, divergent CGIs and promoters to similar degrees. (B) Site Me value of TFAP2C and KISS1R in AGS and AGS treated with 5-aza-dC. In 1400–1800 bp against TSS, the degree of demethylation was high in TFAP2C and low in KISS1R, which representatively showed variation among individual genes. (C) Degree of demethylation of promoters and degree of re-expression in AGS. The degree of demethylation of promoters was not associated with that of re-expression of methylated genes after 5-aza-dC treatment.